Coexisting domains in the plasma membranes of live cells characterized by spin-label ESR spectroscopy.

1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.

Abstract

The importance of membrane-based compartmentalization in eukaryotic cell function has become broadly appreciated, and a number of studies indicate that these eukaryotic cell membranes contain coexisting liquid-ordered (L(o)) and liquid-disordered (L(d)) lipid domains. However, the current evidence for such phase separation is indirect, and so far there has been no direct demonstration of differences in the ordering and dynamics for the lipids in these two types of regions or their relative amounts in the plasma membranes of live cells. In this study, we provide direct evidence for the presence of two different types of lipid populations in the plasma membranes of live cells from four different cell lines by electron spin resonance. Analysis of the electron spin resonance spectra recorded over a range of temperatures, from 5 to 37 degrees C, shows that the spin-labeled phospholipids incorporated experience two types of environments, L(o) and L(d), with distinct order parameters and rotational diffusion coefficients but with some differences among the four cell lines. These results suggest that coexistence of lipid domains that differ significantly in their dynamic order in the plasma membrane is a general phenomenon. The L(o) region is found to be a major component in contrast to a model in which small liquid-ordered lipid rafts exist in a 'sea' of disordered lipids. The results on ordering and dynamics for the live cells are also compared with those from model membranes exhibiting coexisting L(o) and L(d) phases.

Structures of the spin-labeled PCs. The structure shown is that of 5PC, with the doxyl spin label attached to the C5 of the sn-2 acyl chain. Other spin labels, namely 7PC, 10PC, 12PC, and 16PC have the doxyl group attached to C7, C10, C12, and C16, respectively, of the sn-2 acyl chain.

Demonstration of tie-line for SPM/DOPC/Chol. (A) The ternary (compositional) phase diagram of SPM/DOPC/Chol lipid mixtures at 22°C (). It contains an elliptical region of coexisting liquid-ordered (Lo) and liquid-disordered (Ld) phases enclosed by a PB (the elliptical solid line). The short dotted-dashed section at the far left of the PB indicates roughly where there is a possible critical point. The dashed section of the PB to the lower right indicates a region of estimated transition between Lo and Ld phases. The large dots along the PB (1 in Ld phase and 11 in Lo phase) are the compositions at the endpoints of the trial tie-lines and the compositions from which PB spectra were obtained. The solid line with diamonds is the trial tie-line that was determined to be the best estimate to the true tie-line by the method of Chiang et al. (); this line connects the Ld phase with a mol fraction composition of 0.30:0.63:0.07 (Sm/DOPC/Chol) to the Lo phase with composition 0.24:0.42:0.34 (see respective ESR spectra in ). The five diamonds give the compositions on that tie-line whose spectra (spectra 2–6 in ) were fit by a linear combination of the Ld and Lo PB spectra from the end of the tie-line. The other dashed lines are the 10 other trial tie-lines. (B) Demonstrates the differences in the ESR spectra of 16PC at these two boundaries (spectra normalized to a common integrated intensity): dotted line is for Lo, dashed line is for Ld. (C) The solid spectra shown and labeled 2–6 are obtained in the two-phase Lo and Ld coexistence region along the tie-line connecting the two boundary spectra, which are again shown as a dotted line for Lo and a dashed line for Ld. Superimposed on the solid spectra 2–6 are dash-dotted ones that represent the appropriate linear combination of Lo and Ld boundary spectra in accordance with the lever rule () as the compositions are gradually changed from 100% Lo to 100% Ld (from top to bottom), as given by the diamonds along the true tie-line shown in .

ESR spectra of spin labels incorporated into the plasma membrane of live RBL-2H3 mast cells and spectral analysis by the NLLS method. (A) Spectra for 5PC, 7PC, 10PC, 12PC, and 16PC in RBL-2H3 cells recorded at 5°C and 37°C (solid lines) with the corresponding best fit simulations (dotted lines). (B) Spectra for 7PC at 25°C in RBL-2H3, CHO, COS-7, and NIH-3T3 cells. Upper spectra show experimental spectra and fits; middle and lower spectra show the resolved spectra for the more mobile (Ld) and less mobile (Lo) components, respectively. The insets to the upper spectra (×2.5 magnification) show distinctive features of both the Lo and Ld components as marked by vertical arrows.

For RBL-2H3 cells, the best fit values of the order parameter, S0, and the rotational diffusion coefficient, R⊥, of the two components (cross, Lo component; triangle, Ld component) of ESR spectra from different spin labels are plotted versus temperature as solid lines. Also shown are the best fit values of S0 and R⊥ of the two components (circle, Ld component; star, Lo component) of ESR spectra from PMV of RBL-2H3 cells () plotted versus temperature as dashed lines. The thermally averaged fractions of the Ld spectral component for the live cells P(Ld) are 0.26 ± 0.08 (5PC), 0.26 ± 0.04 (7PC), 0.30 ± 0.03 (10PC), and 0.37 ± 0.10 (12PC).

For CHO cells, plots of best fit values of the order parameter, S0, and the rotational diffusion coefficient, R⊥, of the two components (cross, Lo component; triangle, Ld component) of ESR spectra from different spin labels versus temperature. The thermally averaged fractions of the Ld spectral component P(Ld) are 0.25 ± 0.10 (7PC), 0.38 ± 0.02 (10PC), and 0.41 ± 0.11 (12PC). Because of the low signal/noise ratio of the spectra from 5PC in CHO cells, we could not get a good fit for this case; so these results are not provided.

Plots of best fit values of the order parameter, S0, the rotational diffusion coefficient, R⊥, of the two components at 25°C (diamond, Lo component, triangle, Ld component) and (thermally averaged) fraction of the Ld component, P(Ld) of ESR spectra from spin labels 5, 7, 10, and 12PC in RBL-2H3, CHO, COS7, and NIH-3T3 cells, versus the position of nitroxide radical attached at the acyl chain.

ESR spectra of 14PC and 16PC in the plasma membranes of live RBL-2H3, COS7, CHO, and NIH-3T3 cells (experimental, solid lines; simulations, dashed lines). The temperatures at which these spectra were recorded are indicated at the corner of each spectrum. For illustration, the calculated two components of 16PC spectra in COS7 and CHO cells are shown in the two boxes.

Plots of best fit values of the order parameters, S0, and the rotational diffusion coefficient, R⊥, of ESR spectra from spin labels 5, 7, 10, 12, and 16PC in lipid dispersions of SPM/DOPC/Chol with three different compositions (see text) at 22°C versus the position of nitroxide radical attached at the acyl chain, solid lines. Relevant results for RBL-2H3 cells at 25°C from are shown by dashed lines for comparison.

ESR spectra of 7PC in live RBL-2H3 cells at 30°C spectrum 2 (dotted line) was recorded after spectrum 1 (solid line). Both spectra are the sum of 32 repeated scans each. Accumulation of the scans for spectrum 2 was completed 28 min after spectrum 1 was completed. Spectrum 2 was vertically expanded to match spectrum 1 for the purpose of comparison.